In 2009, we generated a soluble protein of the VP8 subunit of VP4 of human rotavirus P genotype 8 (Wa strain), P genotype 4 (DS-1 strain) and P genotype 6 (1076 strain) expressed in E. coli. The combination of these 3 P genotypes constitutes more than 95% of all rotavirus P genotypes detectable worldwide today, which are found in association with G genotypes 1, 2, 3, 4, 5, 8, 9 and 12. Guinea pigs immunized parenterally with a purified Wa VP8 or DS-1 VP8 protein developed high levels of neutralizing antibodies to a corresponding homologous virus and moderate levels of neutralizing antibodies to heterologous DS-1 or Wa strain. The levels of neutralizing antibodies to heterologous P genotype 6 M37 strain were low. Immunization of (i) guinea pigs with a purified 1076 (P genotype 6) VP8 protein;and (ii) mice with a purified Wa VP8 which is conjugated to polysaccharides of P. suis (generated by Drs. Szu and Li of LDMI, NICHS) is in progress. In addition, we expressed successfully in E. coli Wa VP8-EB NSP4 (amino terminal half of the NSP4) fusion protein in a soluble form. Our hope is to use the NSP4 protein (a viral enterotoxin) as an immunostimulating factor to increase the immunogenicity of the recombinant subunit proteins including the VP8. Immunization of guinea pigs with a purified Wa VP8-EB NSP4 fusion protein is underway.